PROJECT SUMMARY In 2010, an estimated 6 million individuals in the United States abused prescription pain relievers, triggering the current opioid epidemic. Further, an estimated 10-20% of women in the U.S. receive a prescription each year for an opioid, such as oxycodone (OXY), during pregnancy. Collectively, this has resulted in a five-fold increase in prescription drug use among expectant mothers over the last decade, as well as one baby born every 15 minutes in opioid withdrawal, termed neonatal abstinence syndrome (NAS). Despite a rapidly growing population of individuals born with NAS, basic research efforts on the effects of prenatal exposure to opioids on brain development, as well as the lifelong behavior impacts, are poorly defined. Better identifying the effects of prenatal OXY exposure on the development of neural circuits and behavior will allow for future investigations into the underlying mechanisms. The long-term goal is development of novel and innovative strategies to mitigate the lifelong impact and the current focus on OXY specifically is based on the perceived safety due to its FDA-approved status. A broad battery of behavioral tests performed in adult mice exposed to OXY in utero indicates this developmental insult produces behavioral deficits related to impulse control and response to opioids, with sex-specific effects, that are consistent with the limited data available on children exposed to opioids in utero. The medial prefrontal cortex (mPFC) is a core member of the neural circuitry governing these behaviors, often with a link to hypofrontality driven by the striatum and amygdala. Thus, the overarching hypothesis is that prenatal OXY alters the development of long-range inputs to the prefrontal cortex, resulting in behavioral dysregulation. To begin addressing this, unbiased monosynaptic circuit tracing was performed in GAD2-Cre mice to create whole brain maps in both sexes of all direct, long-range inputs to mPFC inhibitory neurons. Consistent with the possibility of hypofrontality, this analysis revealed a marked and selective elevation in structural connectivity to mPFC interneurons (INs) from the basolateral amygdala (BLA) in females exposed to prenatal OXY. This led to the working hypothesis to be addressed here, that prenatal OXY exposure produces BLA-mediated inhibition of the mPFC, resulting in behavioral dysregulation. Completion of the two proposed Aims is expected to produce the following: (1) Unbiased whole brain maps of monosynaptic long-range inputs to excitatory and inhibitory (PV, SST and VIP+) mPFC neurons in the context of prenatal OXY exposure, to determine the source and balance of these inputs. (2) Determination of the BLA?s influence over the mPFC following prenatal OXY exposure, in terms of functional connectivity, using high density silicon probes with optogenetic stimulation and behavior, using chemogenetics. The proposed research is expected to provide a framework for future mechanistic studies aimed at further defining the functional subcircuits, how to best mitigate the consequences of maternal opioid use and assessing the impact of current NAS interventions employed in NICUs, which consists of postnatal opioid replacement therapies.